Retinal hypoxia is implicated in the development of many major and common blinding human eye diseases, such as diabetic retinopathy and age-related macular degeneration. The metabolic heath and function of retinal cells is based on the availability of nutrients and oxygen. Two vasculature beds, the blood vessels within the retinal tissue and the choroid, a layer of vascular tissue underneath the retinal tissue, supply oxygen to the retina. Currently, the relative contribution of these vasculatures to the oxygenation of the retinal tissue in health and disease has not been adequately studied, and the role of oxygen in the development of retinal diseases, and their associated vascular pathologies, is not well understood. Therefore, technologies that allow understanding, diagnosis and treatment of diseases in which oxygen is implicated are greatly needed. Although techniques for measurement of blood flow in the large retinal blood vessels are available, they provide only an indirect measure of retinal oxygenation. Invasive methods for retinal oxygen tension measurements that utilize oxygen-sensitive microelectrodes are also available, but not clinically applicable. Recently, measurement of oxygen tension in the retina, with the use of a phosphorescence imaging technique, has been demonstrated. However, due to lack of depth discrimination, this technique does not allow discrete measurement of oxygen tension in the chorio-retinal vasculature and is limited for measurements in the microvasculature of the retina, where the first indication of disease is known to occur. In the proposed research study, we intend to overcome the limitations of the available techniques by establishing a novel system that combines optical section retinal imaging with oxygen-sensitive probe phosphorescence imaging for noninvasive and quantitative differentiation of oxygen tension in the chorio-retinal vasculature of the eye. The specific aims are to develop the system, establish its reliability and reproducibility, determine normal ranges, and detect changes in oxygen tension across the depth of the retinal tissue. Once refined, the system will serve as a valuable tool for three-dimensional retinal oxygen tension imaging. Such imaging will prompt responses to a vast number of eminent research questions on disease-related oxygen dynamics in retinal, choroidal, and intra-retinal vasculatures and thereby, improve diagnosis and understanding of the pathophysiology of retinal diseases.